Many diseases associated with human aging, including cancer (Ohshima et al., Arch. Biochem. Biophys. 2003, 417, 3-11), cardiovascular disorders (Shah et al., Heart 2004, 90, 486-487), and neurodegenerative diseases (Barnham et al., Nat. Rev. Drug Discovery 2004, 3, 205-214; Connor, J. R.; Editor Metals and Oxidative Damage in Neurological Disorders, 1997) have a strong oxidative stress component, but the basic molecular mechanisms that connect aging, age-related diseases, and oxidative stress remain insufficiently understood (Aruoma et al., Editors Molecular Biology of Free Radicals in Human Diseases, 1998; Balaban et al., Cell 2005, 120, 483-495; Finkel et al., Nature 2000, 408, 239-247). Oxidative stress is the result of unregulated production of reactive oxygen species (ROS), and cellular mismanagement of oxidation-reduction chemistry can trigger subsequent oxidative damage to tissue and organs (Beckman et al., Physiol. Rev. 1998, 78, 547-581). In particular, hydrogen peroxide is a major ROS by-product in living organisms and a common marker for oxidative stress. The chemical biology of H2O2 is much more complex, however, as mounting evidence also supports a role for H2O2 as a second messenger in normal cellular signal transduction (Rhee et al., Curr. Opin. Cell Biol. 2005, 17, 183-189; Finkel, T. Curr. Opin. Cell Biol. 2003, 15, 247-254; Stone, Arch. Biochem. Biophys. 2004, 422, 119-124; Wood et al., Science 2003, 300, 650-653). Peroxide bursts in response to cell receptor stimulation can affect several classes of essential signaling proteins that control cell proliferation and/or cell death. Included are kinases like the mitogen-activated protein (MAP) kinase family (Guyton et al., J. Biol. Chem. 1996, 271, 4138-4142), transcription factors such as nuclear factor κB (NF-κB) (Schmidt et al., Chem. Biol. 1995, 2, 13-22), and activating protein 1 (AP-1) (Lo et al., J. Biol. Chem. 1995, 270, 11727-11730) as well as various protein tyrosine phosphatases (PTPs) (Lee et al., J. Biol. Chem. 1998, 273, 15366-15372; Kwon et al., Proc. Nat. Acad. Sci. USA 2004, 101, 16419-16424; Leslie et al., EMBO J. 2003, 22, 5501-5510) ion channels (Avshalumov et al., Proc. Nat. Acad. Sci. USA 2003, 100, 11729-11734; Avshalumov et al., J. Neurosci. 2005, 25, 4222-4231) and G proteins. Despite the far-ranging consequences of H2O2 in human physiology and pathology, mechanistic details surrounding intracellular H2O2 generation, trafficking, and function remain elusive even in the simplest eukaryotic organisms.
Fluorescent probes are well suited to meet the need for tools to map the spatial and temporal distribution of H2O2 within living cells. Such reagents have revolutionized the study of calcium in biological systems and hold much promise for enhancing our understanding of H2O2 physiology and pathology. The major challenge for practical H2O2 sensing in biological environments is creating water-soluble systems that respond to H2O2 selectively over competing cellular ROS such as superoxide (O2−), nitric oxide (NO), and lipid peroxides. Several types of small-molecule reporters have been described for H2O2 detection. Included are dihydro derivatives of common fluorescent dyes (e.g., 2′,7′-dichlorodihydrofluorescein, DCFH, and dihydrorhodamine 123, DHR) (Negre-Salvayre et al., Meth. Enzymol. 2002, 352, 62-71; Hempel et al., Free Rad. Biol. Med. 1999, 27, 146-159; Keston et al., Anal. Biochem. 1965, 11, 1-5; Haugland, R. P. The Handbook: A Guide to Fluorescent Probes and Labeling Technologies; 10th ed.; Invitrogen/Molecular Probes: Carlsbad, Calif., 2005), the Amplex Red/peroxidase system (Zhou et al., Anal. Biochem. 1997, 253, 162-168) phosphine-containing fluorophores (Akasaka et al., Anal. Lett. 1987, 20, 797-807; Onoda et al., Org. Lett. 2003, 5, 1459-1461; Onoda et al., Chem. Commun. 2005, 1848-1850; Soh et al., Bioorg. Med. Chem. 2005, 13, 1131-1139) luminescent lanthanide complexes (Wolfbeis et al., Angew. Chem., Int. Ed. 2002, 41, 4495-4498; Kozhevnikov et al., Inorg. Chim. Acta 2005, 358, 2445-2448 and chromophores with ROS-cleavable protecting groups (Maeda et al., Angew. Chem., Int. Ed. 2004, 43, 2389-2391; Lo et al., Chem. Commun. 2003, 2728-2729; Setsukinai et al., J. Biol. Chem. 2003, 278, 3170-3175). Limitations of these and other currently available H2O2-responsive probes include interfering background fluorescence from competing ROS, potential side reactions with thiols that are present in high concentrations within cells, the need for external activating enzyme, lack of membrane permeability, and/or lack of water solubility or compatibility, requiring the use of organic co-solvents.